Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of linear chains of α-1,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
Sugars from starch, in the form of concentrated dextrose syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (or viscosity reduction) of solid starch with an α-amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA). The resulting syrup has a high glucose content. Much of the glucose syrup that is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup.
α-amylases (EC 3.2.1.1) hydrolyze starch, glycogen, and related polysaccharides by cleaving internal α-1,4-glucosidic bonds at random. This enzyme has a number of important commercial applications in, for example the sugar, brewing, alcohol, and textile industries. α-amylases are isolated from a wide variety of bacterial, fungal, plant and animal sources. The industrially many important α-amylases are those isolated from Bacilli.
For a number of years, α-amylase enzymes have been used for a variety of different purposes, including starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing. These enzymes can also be used to remove starchy stains during dishwashing and laundry washing.
Bacillus licheniformis and other Bacillus species have a high capacity to secrete (heterologous) proteins (e.g., amylases, proteases, etc.) in to the growth medium. To direct these proteins outside the cell, they are synthesized as pre-proteins with an amino-terminal signal peptide. In general, signal peptides, which are usually 18 to 35 amino acids long, do not contain strict consensus sequences. However, the signal peptides do share tripartite structures formed by (1) a positively charged amino terminus (N-domain) and a more polar region, (2) followed by a hydrophobic core (H-domain), and (3) a more polar region, containing the signal peptide cleavage site (C-domain). The amino acids-3 and -1 (relative to the start of the mature protein) are usually residues with small neutral side chains (e.g., alanine, glycine and serine). In B. subtilis, the residue at position 1 of the mature chain is in most cases an alanine (Tjalsma, 1999, “Signal Peptidases of Bacillus subtilis: A Functional Analysis,” Ph.D. thesis, University of Groningen, ISBN 90-367-1086-3). There are observations that indicate that processing of the pre-proteins, i.e. cleavage of the signal peptide, by signal peptidase is a secretion bottleneck for the production of some proteins (Bolhuis, 1999, “A Genetic Analysis of Determinants for Efficient Protein Secretion in Bacillus subtilis,” Ph.D. thesis, University of Groningen, ISBN 90-367-1055-3).
Thus, there is a continued need for α-amylases, wherein greater amounts of α-amylase can be produced in an organism. Increased production will lead to, amongst other things, reduced costs, improved cost margins, plant capacity savings, and higher activity products.